Pathological changes in vessel structure are induced by signaling pathways that govern cell growth, death, differentiation and matrix production. It is postulated that certain heritable forms of cardiovascular diseases result from the dysregulation of cell fate determination programs during ontogeny that predispose to early-onset cardiovascular complications. For example, the CADASIL syndrome (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) is caused by mutations of Notch3. In the parent grant, we hypothesized that Notch3 signaling is a critical determinant of vascular smooth muscle cell (VSMC) fate during vascular remodeling and lesion formation in adults by modulating VSMC growth and apoptosis. Signaling through the Notch receptor is a highly conserved mechanism of cell fate specification that is widely used during embryonic development in both vertebrates and invertebrates. It is postulated that the Notch signaling pathway plays a similar critical role during the embryonic development of the vasculature. The advent of human embryonic stem (hES) cells provides us with an unprecedented opportunity to characterize the role of the Notch3 transcriptional pathway in a model system of human vasculogenesis. Our laboratories have successfully established the use of hES as a model system for studying the developmental biology of human cardiovascular cells. The proposed supplemental project will build upon this technical expertise by testing the central hypothesis that Notch3 signaling is a critical mediator of VSMC differentiation and human vasculogenesis through the sequential activation of an RBPJ-dependent transcriptional pathway in hES cells. Our experimental approach will systematically use genetic probes designed to manipulate the activity of the Notch signaling pathway in order to define its functional role in our hES model. Specifically we will: Aim 1. Define the dynamic spatio-temporal patterns of expression of the Notch3-RBPJ-HRT 1 transcriptional pathway during the process of VSMC differentiation and human vasculogenesis in a human embryonic stem cell model system. Aim 2. Define the role of Notch3-RBPJ-HRT1 transcriptional pathway activation as a sufficient condition for promoting the induction of VSMC fate commitment and vasculogenesis in hES cells. Aim 3. Define the role of Notch3-RBPJ-HRT1 transcriptional pathway activation as a necessary condition for promoting the induction of VSMC fate commitment and vasculogenesis in hES cells. [unreadable] [unreadable]